Worm Breeder's Gazette 7(1): 76
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
We are using immunofluorescence microscopy to screen mouse hybridoma cell lines for the production of monoclonal antibodies against C. elegans antigens. In the course of this work, we observed that the FITC-conjugated rabbit anti- (mouse IgG) used as secondary antibody reacted specifically with cytoplasmic components unique to the germline precursor (P) cells of embryos. Using fluorescent antibody staining, we have followed these components throughout the life cycle. We have termed them P-granules, and the antibody that stains them in P and Z cells we have called PZA. P-granules are detectable in the uncleaved zygote as prelocalized particles at the posterior pole. After the first cleavage they are detected only in the P1 cell. In subsequent divisions they are progressively segregated to the P2, P3, and P4 cells. During these early divisions P-granules become localized prior to cleavage in the region of cytoplasm that is destined for the next P cell daughter. Between the 10-cell and 16-cell stages the number, size, and distribution of P-granules change; the numerous, small cytoplasmic particles present in very early embryos appear to coalesce into larger perinuclear granules. These characteristics of P-granules are similar to those of 'nuage' seen by electron microscopy in early C. elegans embryos. At the 100-cell stage P4 divides into Z2 and Z3, which are the only cells stained by PZA in embryos from the 100-cell stage to hatching of the first stage larva. Within the developing larval gonad, PZA stains perinuclear granules that are seen in all the descendants of Z2 and Z3, but not in the somatic gonad cells. PZA staining of perinuclear granules is also seen in the distal arm of the adult gonad, where germ cells divide mitotically and then enter meiosis. As oocytes mature, the granules disperse from the nuclei. PZA stains mature oocytes diffusely; granules are sometimes observed randomly distributed in the cytoplasm. Mature sperm obtained from males show cytoplasmic staining by PZA. There is also a high level of non-gonadal staining in late larvae and adults. The cross-reactivity of PZA seems to be limited. We have tested embryos of mouse, Drosophila melanogaster, d Panagrellus redivivus. Only the Panagrellus embryos showed specific staining of what are presumably the germline precursor cells. Immunofluorescent staining of P-granules has been observed with 3 different lots of fluorochrome-conjugated rabbit anti-mouse antibody ( F-RAM) from Miles Laboratories, as well as 2 lots of fluorochrome- conjugated goat anti-rabbit antibody (F-GAR) from Miles. However, F- RAM and F-GAR from 2 other companies do not stain P-granules. The Miles F-RAM used in this study was prepared from the pooled sera of 15 rabbits that had been immunized with mouse IgG. Some possible explanations for the presence of PZA in the F-RAM are: 1) PZA may be a rabbit autoantibody to an evolutionarily conserved or cross-reacting rabbit antigen. 2) PZA may have been elicited by a contaminant in the mouse IgG injected into the rabbits as immunogen. 3) One or more of the immunized rabbits may have had a nematode infection, which elicited production of antibodies, including PZA, that cross-react with C. elegans. The third explanation is consistent with both the limited cross-reactivity of PZA with other species and the high level of general staining of larval and adult C. elegans preparations by Miles F-RAM. We recently tested the serum from 11 wild and potentially worm-infected rabbits from a local farm; screening by indirect immunofluorescence, we found that 1 of the 11 serum samples contained PZA. We are presently using the Miles F-RAM and F-GAR to ask about the composition, mechanism of segregation, and possible role of P-granules. The wild PZA-producing rabbit may help us determine the origin of the antibody.